Project 1 of this multi-project application will focus on the development of rapid membrane tests (dipsticks) Project 1 of this multi-project application will focus on the development of rapid membrane tests (dipsticks)for Entamoeba histolytica, Giardia, Cryptosporidium, and shiga toxin from Shigella spp/enterohemorrhagic Escherichia coli. These parasites are Category B pathogens that are global healthcare problems. We will focus on developing immunogold-based tests that will be more robust than assays that use enzyme-tagged antibodies. Individual tests for each parasite, as well as a multi-component lateral flow syndromic test will be developed. Although these tests are being developed in response to the biodefense initiative, they will be robust enough for use in developing countries. We have developed immunogold-based dipsticks for E. histolytica that meet this criteria by being highly stable, rapid and simple to use. Our work on immunogold-based dipsticks will be extended to include Giardia and Cryptosporidium. In addition, our work on the E. histolytica dipsticks will be extended for the detection of circulating antigen in persons with amebic liver abscess. As a natural extension of this work, we will utilize a new platform technology that employs antibody-tagged paramagnetic particles for increased sensitivity. This new technology will be used to develop a dipstick for shiga toxin that is similar in sensitivity to tissue culture cytotoxicity assay. As a long term goal, a multi-component syndromic test panel employing antibody-tagged paramagnetic particles will be developed for E. histolytica, Giardia, Cryptosporidium, and shiga toxin. The test will be rapid, simple to use, and highly sensitive. We will transfer the technology to a manufacturer of rapid membrane tests and establish test performance of dipsticks manufactured under GMP. Data for 510(k) submissions to the Food and Drug Administration will be collected from in-house performance studies and on-site clinical evaluations that utilize PCR technology for critical test evaluation. All of the tests developed in Project 1 will be critically evaluated through comparison with PCR at on-site locations. For developing world applications we will explore transfer of dipstick technology to local manufacturers to ensure efficient, large-scale, and low-cost production. These tests will have applicability as point-of-care tests for the diagnosis of individuals exposed to Category B pathogens.